Instrument for independent electrotransfer in multiple cassettes

ABSTRACT

Electrotransfer is performed in an instrument that receives electroblotting cassettes and that contains an integrated power supply, controls, and a display that allows the user to monitor and control each of a plurality of cassettes individually through electrical contacts within the housing that mate with corresponding electrical contacts on the cassettes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/963,430, filed on Dec. 8, 2010, which claims the benefit of U.S.Provisional Patent Application No. 61/285,661, filed Dec. 11, 2009, thecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention resides in the field of gel electrophoresis, and relatesin particular to the transfer of electrophoretically separated speciesfrom a slab gel or any other two-dimensional medium in which the specieswere separated to a two-dimensional support matrix in which the speciescan be detected, identified, and quantified.

DESCRIPTION OF THE PRIOR ART

Proteins, nucleic acids, or other biological species that have beenelectrophoretically separated in a slab gel are often transferred to amembrane of nitrocellulose, nylon, polyvinyl difluoride, or similarmaterials for identification and quantification which are more easilyperformed on the membrane than in the gel. A common transfer techniqueis electroblotting, in which the flat surfaces of the gel and membraneare placed in direct contact and an electric current is passed throughboth the gel and the membrane in a transverse direction, therebytransferring the species in a manner similar to that by which thespecies were mobilized within the gel. When the species are DNAfragments, the transfer is termed a Southern blot after its originator,the British biologist Edwin M. Southern. By analogy, the transfer of RNAfragments is termed Northern blotting, and the transfer of proteins orpolypeptides is termed Western blotting. Once transfer has occurred, thespecies on the membrane are analyzed by methods appropriate to thespecies themselves. In Southern and Northern blots, for example, theanalysis involves treatment of the species on the membrane with ahybridization probe, followed by labeling them with a fluorescent orchromogenic dye. In Western blots, the species are treated withantibodies, followed by the use of conventional labeling techniques todetect the antibodies.

Electroblotting of the Southern, Northern, and Western types can all beperformed in either a wet, dry, or semi-dry format. In wet blotting, thegel and membrane are layered over each other in a stack which isimmersed in a transfer buffer solution in a tank on whose walls aremounted wire or plate electrodes. The electrodes are then energized tocause the solutes to migrate from the gel to the membrane. In semi-dryblotting, filter papers wetted with the transfer buffer solution areused, and the stack contains the filter papers on the top and bottomwith the gel and the membrane between the filter papers to form a“blotting sandwich.” The electrodes are then placed in direct contactwith the exposed surfaces of the wetted filter papers. Dryelectroblotting uses no liquid buffers other than those residing in thegels. Descriptions of wet, dry, and semi-dry electroblotting and theassociated materials and equipment are found in Margalit et al.(Invitrogen) United States Patent Application Publication Nos. US2006/0272946 A1 , published Dec. 7, 2006, US 2006/0278531 A1, publishedDec. 14, 2006, and US 2009/0026079 A1, published Jan. 29, 2009;Littlehales (American Bionetics) U.S. Pat. No. 4,840,714, issued Jun.20, 1989; Dyson et al. (Amersham International) U.S. Pat. No. 4,889,606,issued Dec. 26, 1989; Schuette (Life Technologies, Inc.), U.S. Pat. No.5,013,420, issued May 7, 1991; Chan et al. (Abbott Laboratories), U.S.Pat. No. 5,356,772, issued Oct. 18, 1994; Camacho (Hoefer ScientificInstruments), U.S. Pat. No. 5,445,723, issued Aug. 29, 2005; Boquet(Bertin & Cie), U.S. Pat. No. 5,482,613, issued Jan. 9, 1996; and Chen(Wealtec Enterprise Co., Ltd.) U.S. Pat. No. 6,592,734, issued Jul. 15,2003.

SUMMARY OF THE INVENTION

The present invention resides in an instrument that can performelectrotransfer in either a single electrotransfer cassette or in two ormore such cassettes simultaneously and independently. The term“electrotransfer cassette” is used herein to mean any receptacle thatcontains electrodes and can accommodate a gel or other medium that haschemical or biological species distributed therein in a two-dimensionalarray such as the wells of a microtiter plate, plus a membrane or othertwo-dimensional matrix to which the species are to be transferred by theinfluence of the electric field generated by the electrodes. Theinstrument is particularly suited to cassettes that are designed toperform electroblotting from slab gels. The invention is also wellsuited to cassettes that have electrical contacts on their exteriorsurfaces that are electrically connected to the electrodes inside thecassettes. The instrument contains a power supply and uses batterycontact technology to connect the power supply with each cassette,thereby allowing the instrument to be used either with its full capacityof cassettes or with only one cassette or a number of cassettes lessthan the full capacity of the instrument, utilizing only the powerneeded for the cassette(s) present in the instrument. The battery-styleelectrical contacts are mounted in the interior of the instrument andinaccessible to the user even when the instrument is empty or has anunoccupied space, thereby avoiding inadvertent exposure of the user toan electric current. Connection of a cassette to the instrument contactsis achieved by simple insertion of the cassette in the instrument, andin certain embodiments of the invention the instrument containsautomatic relays that block the power supply to the electrical contactsfor each individual cassette when the cassette is removed. In preferreddesigns, the instrument holds multiple cassettes in a vertical stack,thereby providing the instrument with a small footprint that consumes aminimum of bench space. Further features of the instrument will beapparent from the descriptions that follow and the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an instrument in accordance with thepresent invention that accommodates two cassettes.

FIG. 2 is a perspective view of the instrument of FIG. 1 with one of thetwo cassettes fully inserted and another partially inserted.

FIG. 3 is a cutaway view of the instrument of FIG. 1.

FIG. 4 is an enlarged view of one set of electrical interconnects in theinterior of the instrument of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

With electroblotting cassettes as illustrative examples, electroblottingcassettes that can be used with the instrument of the present inventionare described in commonly owned U.S. Provisional Patent Application No.61/285,277, filed Dec. 10, 2009, entitled “Electroblotting Cassette WithIntegrated Electrical Contacts and Rotary Locking Mechanism” (M. Latham,inventor), and United States Pre-Grant Patent Publication No. US2010-0213064 A1, published Aug. 26, 2010, entitled “ElectroblottingCassette With Manually Releasable Electrodes of Adjustable Spacing” (M.Latham, inventor). In general, the instrument will receive anyelectrotransfer cassette that contains external electrical contacts.

The instrument 11 shown in FIGS. 1 and 2 holds two such cassettes 12, 13in a vertical stack. Both are inserted independently by a slidinginsertion through slot-shaped openings in the front of the instrument,or through a single opening with guide rails at different heights alongthe internal walls of the instrument. The lower cassette in FIG. 2 isshown partially inserted for purposes of demonstration. Instrumentswithin the scope of the invention can be constructed to accommodate anynumber of cassettes, and instruments that accommodate two, three, four,five, or six cassettes will most likely be the most common. Thereceiving slots or positions can be arranged vertically as shown orhorizontally, or in two-dimensional arrays forming columns and rows. Theinstrument can be operated with cassettes occupying all slots orpositions or with one or more slots or positions left unoccupied andtherefore unused. The electronics are designed to allow independentcontrol over individual positions and the cassettes occupying thosepositions.

The front of the instrument contains a display 14 showing the conditionsof the electroblotting procedure for each cassette, including timing,voltage, current, and running parameters in general, and optionallyadditional parameters such as indicators to show the number of insertedcassettes detected by the instrument and failure diagnostics. Thedisplay can be a touch screen with incorporated programming controls forindividual cassettes. Alternatively, the programming controls can beincorporated in a membrane keypad overlying the display and the adjacentsurface of the instrument housing, or a keypad incorporated into thehousing itself.

FIG. 3 shows the instrument housing 21 in a cutaway view, with the uppercassette 12 fully inserted and the lower cassette 13 partially insertedas in FIG. 2. Mounted inside the housing 21 are a printed circuit board22 and a power supply 23. The power supply 23 can be a commoncommercially available component such as one with universal input and a30V dc output. The dc output is directed to amplifiers on the board 22that control the voltage to each cassette. The amplifiers and othercomponents on the board preferably form a multitude of independentelectrical channels, one for each cassette slot. A fan 24 mounted in therear wall of the housing (only the mounting frame for the fan is shown)cools the electronic components. The power supply 23 and the board 22 inthis embodiment are positioned above the upper cassette slot in astacked arrangement that preserves the small footprint of the instrumentand thereby conversed bench space.

FIG. 4 is an enlarged view of a portion of the internal surface of therear wall of the instrument housing. Mounted to this wall are a pair ofslugs 31, 32 of magnetically responsive material such as steel. Theslugs are aligned with magnets on the rear of each cassette when thecassette is inserted in the instrument, and the slugs and magnets serveto secure the inserted cassette in place. Reed relays are alsoincorporated into the housing in the region of these slugs to controlthe activation of the electronics for individual cassette slots uponinsertion or removal of a cassette, thereby blocking the power supply tothe electrical contacts at each position that is not occupied by acassette. Any relay that can be activated by insertion of the cassetteand deactivated upon removal of the cassette can be used.

Also visible in FIG. 4 are the electrical contacts 33, 34, 35, 36 thatsupply voltage to the cassette when the cassette is inserted bycontacting the electrical contacts on the back of each cassette. Of themany types of contacts that can be used, an example are battery-stylecontacts, i.e., spring-loaded contacts known in the art as “pogo pins.”Voltage is supplied to the contacts by the amplifiers on the board 22.In the embodiment shown, two electrical contacts are included for eachelectrode in the cassette: the two central pins 34, 35 will engage thesingle anode contact on the cassette while the two outer pins 33, 36will engage the two cathode contacts on the cassette that flank theanode contact. A single contact for each electrode will also suffice,although the redundancy of multiple contacts is preferred for a morereliable connection. The number and positions of these pins will bevaried to correspond to any variations in the number and spatialarrangement of the contacts on the cassette. The pins are mounted insiderigid sleeves 41, 42, 43, 44, which serve as stops for the cassette andprevent contact between the back end surface of the cassette and therear wall of the housing. Raised bosses 45, 46 surrounding the steelslugs serve a similar function. The housing can also contain horizontalgrooves in the internal surfaces of the side walls of the cassette slotsto mate with ridges on the side edges of the cassettes, or vice versa,to help guide the cassettes into the slots. The grooves can eliminatethe need for individual slots for the cassettes, and instead allow thehousing to be constructed with a single large opening to accommodate thefull number of cassettes in a stacked arrangement, with grooves arrangedat multiple heights within the opening to position the individualcassettes and to align them with the electronic interconnects at theinner rear wall of the housing.

To utilize an instrument in accordance with the above descriptions inelectroblotting procedures for transferring electrophoreticallyseparated species from a slab gel to a sheet-form matrix such as amembrane of nitrocellulose, nylon, polyvinyl difluoride, or othermaterial on which treatments or analysis are typically performed in abiochemical laboratory, the gel and membrane, typically in combinationwith buffer-wetted filter papers, are first arranged in a stack andplaced in a cassette which includes plate electrodes as the anode andcathode plus external electrical contacts. The cassette, together withfurther cassettes in many cases, is then placed in the instrument, andthe instrument is programmed to impose electrical charges on theelectrodes to produce an electric field transverse to the planes of thegel and membrane sufficient to cause the species to migrate to themembrane. As explained above, a single cassette can be processed in thismanner, or two or more cassettes simultaneously in the same instrument,either under identical or individually controlled conditions.

Further alternatives to the structures, shapes, and arrangements shownin the figures that are still within the concept of the presentinvention include electrical contacts other than the Pogo pins shown inthe Figures, but still either spring-loaded like the Pogo pins, or otherresilient or non-resilient configurations that will ensure properelectrical contact. Similarly, electrical actuators other than Reedrelays that will shut off power when a cassette is removed can be used,and other variations can be made that will be readily apparent to thoseskilled in the art.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is not excluded from the scope of the claim. All patents,patent applications, and other published reference materials cited inthis specification are hereby incorporated herein by reference in theirentirety. Any discrepancy between any reference material cited herein orany prior art in general and an explicit teaching of this specificationis intended to be resolved in favor of the teaching in thisspecification. This includes any discrepancy between an art-understooddefinition of a word or phrase and a definition explicitly provided inthis specification of the same word or phrase.

What is claimed is:
 1. An electrotransfer instrument comprising: ahousing with an internal cavity sized to contain an electroblottingcassette, said cavity mating with ridges on the side of said cassette; apower supply and printed circuit board mounted to and within saidhousing and individual electrical contacts mounted to the interior ofsaid housing for connecting said power source via the electricalcontacts to electrodes in said cassette that is inserted therein; and atouch screen display for independently monitoring and controlling anelectrotransfer procedure in said cassette inserted in said housing. 2.The electrotransfer instrument of claim 1 wherein said housing comprisesa front opening for sliding entry of said cassettes.
 3. Theelectrotransfer instrument of claim 1 wherein said housing comprisesmagnetic contacts to secure individual cassettes within said internalcavity.
 4. The electrotransfer instrument of claim 1 further comprisingan electrical relay that will engage said electrical contacts with saidpower source only when a cassette is inserted in said housing.
 5. Theelectrotransfer instrument of claim 1 wherein said cassette furthercomprises a gel, a membrane, and buffer-wetted filter papers arranged ina stack.
 6. The electrotransfer instrument of claim 5 wherein saidcassette further comprises plawte electrodes arranged in a stack, whichare connected to said electrical contacts.
 7. The electrotransferinstrument of claim 1 wherein said individual electrical contacts arespring-loaded electrical contacts.
 8. A method for transferringelectrophoretically separated species from a slab gel to a sheet-formmatrix, said method comprising: (a) placing said slab gel and asheet-form matrix in an electroblotting cassette that comprises anodeand cathode plates and external electrical contacts for each of saidplates; (b) inserting said cassette in the electrotransfer instrument ofclaim 1 such that said external electrical contacts are in contact withsaid individual electrical contacts; and (c) imposing electrical chargeson said electrodes to cause said species to migrate electrophoreticallyfrom said slab gel to said sheet-form matrix within said insertedcassette.